The most commonly used process in nucleic acid synthesis employing solid phase chemistries is the phosphoramidite approach. 5′-Hydroxynucleotides, bound to a polymeric carrier, is phosphitylated with 3′-(β-cyanoethyl-N,N-diisopropylphosphoramidite)nucleosides activated by protonation with weak acids. Acids and amidites are used in excess relatively to the bound nucleotides to ensure a rapid and complete formation of the internucleotide phosphotriester linkage. Phosphoramidite reagents have been activated by addition of tetrazole (pKa 4.9), dicyanoimidazole (pKa 5.2), or pyridinium salts (pKa=5.2-5.5). Other activating agents has been used such as 5-trifluoromethyl-1H-tetrazole, 5-ethylthio-1-H-tetrazole, 5-benzylthio-1-H-tetrazole, 2,4,5-tribromoimidazole, 2-nitroimidazole, 1-hydroxy-benzotriazole, 5-chlorobenzotriazole, and benzimidazolium, imidazolium, pyridinium, N-methylimidazolium, N-methylanilinium trichloroacetate salts. References related to those activators can be found in Eleueri et al., Organic Process Research & Development 2000 and U.S. Pat. No. 6,642,373.
In automated nucleic acid synthesizers, amidite and activator solutions are delivered successively to the solid supports. Poor mixing of the two solutions lead to lower phosphitylation yields and a growing accumulation of sequence failures by failing to activate the amidites. Trends towards higher throughput and lower synthesis scale (i.e., 1 to 20 nmols) accentuate this drawback as smaller volume of reagents are being delivered to the solid supports. To minimize or eliminate these drawbacks, we anchored activator moieties to the polymeric carriers commonly used in the solid phase synthesis of nucleic acids. The said activators have pkas in the 4 to 7 range and are being reprotonated (i.e. regenerated) during each detritylation step, prior to the phosphitylation step (vide infra).